Autophagy initiation triggers p150Glued–AP-2β interaction on the lysosomes and facilitates their transport

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued–AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued–AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued–AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued–AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued–AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05256-6.

and grown in Dulbecco's modified Eagle's medium (DMEM) with 4500 mg/ml of D-glucose that contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Sigma-Aldrich) at 37°C in a 5% CO2 atmosphere.For the proximity ligation assay (PLA) and PLAelectron microscopy (EM) experiments, Rat2 cells were grown on glass coverslips or Thermanox plastic coverslips (Thermo Fisher; catalog no.174950), respectively, coated with 0.2% gelatin for 1 h at 37°C.For live experiments, Rat2 cells were seeded on glass coverslips that were coated with poly-L-lysine (50 μg/ml in H2O for 1 h).For plasmid DNA transfection, HEK293T cells at 70% confluency were transfected using polyethylenimine PEI 25K (Polysciences, catalog no.23966) according to the manufacturer's protocols.After transfection, HEK293T cells were grown in DMEM that was supplemented with 5% FBS for 48 h.Rat2 cells were transfected with plasmid DNA using electroporation.For each transfection, a total of 10 6 cells were suspended in Opti-MEM medium (Thermo Fisher, catalog no.31985-047), mixed with 10 μg of DNA, and added to 2 mm gap cuvettes (Nepagene, catalog no.EC-002S).Cells were electroporated using a NEPA21 electroporator (Nepagene) with poring pulses (6, 150 V, 2.5 ms length, 50 ms interval, 10% decay rate), followed by transfer pulses (5, 20 V, 50 ms length, 50 ms interval, five pulses, 40% decay rate) with "±" polarity set for both.After electroporation, the cells were grown in DMEM with 10% FBS without antibiotics.The medium was changed the next day for a medium that contained 1% penicillin-streptomycin.
The siRNA transfection of HEK293T and Rat2 cells was performed on trypsinized cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher, catalog no.13778150) according to the manufacturer's protocol.For the combined siRNA transfection/DNA electroporation experiments, cells after siRNA transfection were seeded on 10 cm plates and allowed to attach.After 24 h, cells were harvested by trypsinization and electroporated as described earlier.After electroporation, each variant was seeded on glass coverslips that were coated with poly-L-lysine (50 μg/ml in H2O for 1 h).

Primary neuron preparation and transfection
Primary hippocampal cultures were prepared from embryonic day 18 rat brains as described previously [1].The rats that were used to obtain neurons for further experiments were sacrificed according to protocols that complied with European Community Council Directive 2010/63/EU.Transfections of hippocampal neurons were performed on day in vitro 5 (DIV5) with Lipofectamine2000 (Thermo Fisher, catalog no.11668019) as described previously [1], except that the incubation time with the transfection mixture was reduced to 2 h.DNA (2 µg) and 1.5 µl of Lipofectamine2000 were used per well of a 12-well dish.

Proximity ligation assay
For the PLA, cells were fixed for 5 min with ice-cold 100% methanol and 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at -20°C, followed by 10 min with 4% paraformaldehyde (PFA)/4% sucrose in phosphate-buffer (pH 7.4).Fixed cells were washed three times with phosphate-buffered saline (PBS) and incubated with mouse anti-p150 Glued and rabbit anti-AP-2β antibodies that were diluted in PBS that contained 1% donkey serum and 0.2% Triton-X100 at 4°C overnight.The next day, the cells were washed twice for 5 min with PBS with 1% donkey serum and 0.2% Triton-X100, washed once for 1 min in antibody diluent that was provided in the manufacturer's PLA kit (Sigma-Aldrich, catalog no.DUO92101), and then incubated for 60 min at 37°C with relevant secondary antibodies that were conjugated to oligonucleotide PLUS or MINUS (Sigma-Aldrich; catalog no.DUO92002 and DUO92004, respectively) and diluted in antibody diluent.The coverslips were then washed twice for 5 min with buffer A (Sigma-Aldrich, catalog no.DUO82046).
Ligation and amplification were performed according to the manufacturer's protocol using Duolink In Situ Detection Reagents Red (Sigma-Aldrich, catalog no.DUO92008).The cells were then counterstained with mouse anti-tubulin antibody that was diluted in PBS that contained 1% donkey serum and 0.2% Triton-X100 for 30 min at room temperature and washed three times with PBS and incubated with anti-mouse Alexa-488-conjugated secondary antibody that was diluted in PBS that contained 1% donkey serum and 0.2% Triton-X100 for 30 min at room temperature to visualize cell shape.Finally, the cells were washed three times with PBS and mounted with DuoLink in situ mounting medium that contained DAPI (Sigma-Aldrich, catalog no.DUO82040).For each experiment, negative controls that lacked one of the primary antibodies were used.

Immunofluorescence
For analyses of the co-localization of p150 Glued and AP-2β proteins, Rat2 cells were fixed with ice-cold 100% methanol with 2 mM EGTA for 5 min at -20°C and then with 4% PFA/4% sucrose in phosphate-buffer (pH 7.4) for 10 min at room temperature and washed three times with PBS.After fixation, the cells were blocked for 1 h in blocking buffer (5% donkey serum, 0.3% Triton X-1001 in PBS).Next, the cells were incubated overnight with primary antibodies in antibody dilution buffer (1% BSA, 0.3% Triton X-100 in PBS) at 4°C and then washed three times with PBS at room temperature.Afterward, specimens were incubated with Alexa 488-and Alexa 594-conjugated secondary antibodies for 1 h at room temperature, followed by three washes with PBS.Coverslips were mounted with Prolong Gold with DAPI (Thermo Fisher, catalog no.P36941).For the immunofluorescent staining of ribosomal protein S6 phosphorylated at Ser235/236 (P-S6), neurons were fixed with 4% PFA/4% sucrose in phosphate-buffer (pH 7.4) for 10 min at room temperature and washed three times with PBS.
After fixation, the cells were blocked for 1 h in blocking buffer (5% donkey serum, 0.3% Triton X-1001 in PBS).Next, the cells were incubated overnight with primary antibody in antibody dilution buffer I (1% BSA, 0.3% Triton X-100 in PBS) at 4°C and then washed three times with PBS at room temperature.Alexa 488-conjugated secondary antibody with Alexa 647conjugated phalloidin (1:1000; Thermo Fisher Scientific) in antibody dilution buffer was next added for 1 h at room temperature.Afterward, the cells were washed three times with PBS, and coverslips were mounted with Prolong Gold (Thermo Fisher, catalog no.P36934).For the immunofluorescent detection of CLIP-170, the cells were fixed for 5 min with ice-cold 100% methanol and 2 mM EGTA at -20°C, followed by 10 min with 4% PFA/4% sucrose in phosphate-buffer (pH 7.4) at room temperature, and then washed three times with PBS.Next, the cells were washed twice with antibody dilution buffer II (1% donkey serum, 0.2% Triton X-100 in PBS) and incubated overnight with the primary antibody in antibody dilution buffer II at 4°C.After incubation with primary antibodies, the cells were washed three times with PBS at room temperature and incubated with Alexa 488-conjugated secondary antibody in antibody dilution buffer II for 1 h at room temperature.Afterward, the cells were washed three times with PBS, and nuclei were stained with Hoechst 33258 (1 µg/ml; Thermo Fisher Scientific) for 5 min.The cells were then washed two more times with PBS, and coverslips were mounted with Prolong Gold.To analyze LC3B immunofluorescence, the cells were fixed according to the CLIP-170 staining protocol.After fixation, the cells were blocked for 15 min at room temperature in antibody dilution buffer III (2% donkey serum, 0.2% Triton X-100 in PBS) and incubated for 1 h with the primary antibody in antibody dilution buffer III at room temperature.
The cells were then washed three times with antibody dilution buffer III at room temperature and incubated with Alexa 488-conjugated secondary antibody in antibody dilution buffer III for 1 h at room temperature.The next steps were identical to the description of CLIP-170 immunofluorescence.

Fixed cell image acquisition and analysis
Microscopic images of fluorescently labeled in vitro cultured cells, with the exception of quantitative co-localization experiments, were acquired using a Zeiss LSM800 confocal microscope (40 or 63 Plan Apo oil immersion objective, NA=1.4) as Z-stacks at 0.2-0.5 μm intervals at 1024  1024 pixel resolution.To obtain higher image resolution and conduct studies of protein co-localization, an AiryScan detector was used with the 63 oil objective.To enable comparisons between images, the microscope settings were kept constant for all scans.The Zstacks, with the exception of images acquired in AiryScan mode, were converted to single images using a maximum intensity projection.For quantification of the number of PLA puncta in Rat2 cells, the cell shape was indicated as a region of interest for each cell, and the threshold was set manually and uniformly for all images in each experiment to extract specific signals from background.The number of PLA puncta per cell was counted in ImageJ software using the "Analyze Particles" macro.The average number of PLA puncta per cell was then calculated for each condition, and the results were normalized by subtracting the average number of PLA puncta per cell from the negative control.The obtained average number of PLA puncta per cell was further normalized by dividing the obtained values from a particular experiment by the average number of PLA puncta per cell from the control from at least three repetitions.For the analysis of P-S6 levels in neurons, the mean fluorescent intensity of P-S6 was measured in the neuronal soma using ImageJ software.
For quantitative co-localization analysis, images of fluorescently labeled cells were registered using a Leica SP8 confocal microscope with a 63 oil immersion Plan Apo objective lens (NA=1.4).The microscope system was equipped with acousto-optical beam splitter and multialkali single-channel PMTs and hybrid detectors (HyD).The fluorescence of DAPI was excited with a 405 nm light (5 mW diode laser) and detected in the 410-470 nm range.The fluorescence of Alexa 488 and Alexa 568 was excited with a supercontinuum (white light) laser (15 mW) at their absorption maxima and detected at 490-550 nm and 575-630 nm ranges, respectively.The images (optical sections) were registered using PMT (gain 600, DAPI) or HyD (gain 200, Alexa) working in the integration mode at 16-bit precision.The pixel size was 90 nm in the x-y plane and 350 nm along the optical axis.The pixel dwell time was 2.5 µs (4 averaging), and the confocal pinhole was set to 1 Airy unit (at 530 nm).The cells were imaged at room temperature.Stacks of optical sections (Alexa 488 and Alexa 568) were subjected to blind deconvolution (15 iterations) using Huyghens v. 3.7 (Scientific Volume Imaging, Hilversum, The Netherlands).Processing was initialized with the nominal PSF of the objective and performed at an SNR set to 15.The background was estimated as a minimum of average intensity in a 25  25 region.Following deconvolution, the nuclei (DAPI) were segmented using Otsu thresholding (two classes).Overlapping nuclei were separated by Euclidean distance transform, followed by watershedding.The Alexa 488 and Alexa 568 images were summed, processed with a median filter (7  7  3 size) and dilated (11  11  3 neighborhood).The cells were segmented using Otsu thresholding, and single cell masks were constructed with iterative dilation of the nuclear masks.The dilation steps were ordered by the intensity of processed cell images.Volumes that corresponded to nuclei were then excluded from the cell masks.

Coefficients of intensity correlation (Pearson and Spearman) between Alexa 488 and Alexa 568
images were calculated on a cell-by-cell basis using the respective masks.The operation was repeated using the Alexa 568 images that were shifted by ±8 voxels in the x and y directions and ±2 voxels in the z direction.The respective coefficients were calculated using the union of two respective cellular masks and corresponded to the random association of Alexa 488 and Alexa 568 fluorescence in a cell.The raw correlation coefficients were then divided by their random counterparts (on a cell-by-cell basis) to create standardized Pearson and Spearman values.

Analysis of PLA signal co-occurrence with Lamp-1-GFP
Analysis of the proximity of the PLA dots to the LAMP1-GFP vesicles was performed using 16-bit microscopic images acquired with the LSM800 Airyscan module.Only the middle optical section from 5-step z-stack was used for the analysis.The analysis was performed with the ImageJ software.First, the signal of a channel containing PLA puncta was converted into a binary mask using a manually selected threshold for each photo.Then, the captured binary points were expanded using the "Dilate" command to enable the detection of events where the signals touch but do not directly overlap.Based on these points, regions of interest (ROIs) were created in which the fluorescent signal of the GFP channel was measured using the mean gray value.Finally, calculations were performed in which points were labeled as co-occurring with Lamp1-GFP if the mean fluorescence signal of GFP in the ROI was above the established threshold of 1000 pixel brightness value.

Live imaging of microtubule dynamics
For microtubule dynamics analysis, Rat2 cells were electroporated with pEGFP-EB3 or pEGFP-CLIP-170 plasmids.Forty-eight hours later, time-lapse movies of EGFP-EB3 or EGFP-CLIP-170 comets were taken using an Andor Revolutions XD spinning disc microscope with a 63x objective and 1.6 x tube lens (optovar) at 1004 x 1002 pixel resolution.
Images were taken with an exposure of 200 ms and interval of 0.3 s, collecting a total of 600 frames over 3 min.During imaging, cells were kept in a Chamlide magnetic chamber (Quorum Technologies) at 37°C with 5% CO2 in the incubator that was part of the microscope system.
Only EGFP-EB3 or EGFP-CLIP-170 comets whose movements lasted at least four consecutive frames and had a displacement length of at least 10 pixels (0.68 µm) were analyzed using the ImageJ "TrackMate" plugin.The reported values are the number of tracks (i.e., the quantification of objects that were detected as comets), the total run length of comets before catastrophe (Track Displacement), comet lifetime (Track Duration), and Track Mean Speed.
For the analysis, values were calculated as means for each cell.

Opera Phenix High content imaging of fixed cells
For the high-throughput analysis of effects of different drugs on lysosomal acidity, Rat2 cells were seeded on CellCarrier-96 well Black glass bottom plates (Perkin Elmer) that were coated with 0.2% gelatin at a density of 7  10 3 cells per well 1 day before treatment (see above).After 80 min of drug exposure, LysoTracker Red DND-99 (500 nM, Invitrogen, catalog no.L7528) was added to the cells for the last 40 min.Subsequently, the cells were fixed with 4% PFA for 10 min and subjected to three 10-min washes with PBS.High-content screening microscopy was conducted using the Opera Phenix system (PerkinElmer) that was equipped with a 40 1.1 NA water immersion objective.Image acquisition and subsequent analysis were performed using Harmony 4.9 software (PerkinElmer).Statistical analyses were performed using the R and RStudio software packages (Cran).To ensure consistent and reliable data analysis, cells were initially filtered based on morphological criteria to obtain uniform cell populations.The identification of lysotracker-positive compartments was achieved using the "Find spots" algorithm that is integrated within the Harmony software.Additionally, the total signal intensity of Lysotracker staining was quantified within both the cell cytoplasm and the previously defined compartment regions.

Protein extraction from adult rat brains and immunoprecipitation
For the isolation of endogenous proteins from brain extracts, Dynabeads™ Protein G resin conjugated to the primary antibody recognizing the selected protein was prepared.For this purpose, 30 μl of the resin was incubated with 1 μg of the antibody for 1 hour at room temperature.A non-specific IgG antibody from the same animal species as the respective primary antibody was used as a negative control.Rat cortex extract was obtained by homogenizing the tissue in lysis buffer containing 40 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5 mM CaCl2, 0.3 % CHAPS, protease and phosphatase inhibitors.In case of rapamycin-treated variant, 100 nM rapamycin was added to the lysis buffer for brains of rapamycin-treated animals.The homogenate was incubated for 1 hour at 4 °C, and then centrifuged (15 minutes, 4 °C, 18,000 x g) to remove cell debris.The protein concentration in the filtrate was then determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific; catalog no.23225).A portion of the clear protein extract at 3 mg/ml was retained as the starting fraction ("input"), and the remainder was applied to antibody-conjugated beds and incubated overnight at 4 °C.The next day, the beds were washed four times with a wash buffer consisting of 40 mM HEPES, pH 7.5; 300 mM NaCl; 1 mM MgCl2; 0.5 mM CaCl2; 0.1 % CHAPS.After the last wash, the bed with the bound proteins was resuspended in Laemmli buffer and heated at 96 °C for 5 minutes.The prepared samples were analyzed using the Western blot technique.

Protein production in bacteria and pull-down experiment
Recombinant proteins were produced in the E. coli BL21 strain.Individual clones that were transformed with plasmids that encoded proteins of interest were picked from the plates and inoculated to 5 ml of LB with appropriate antibiotic.After overnight culture, at 37°C with shaking, bacteria were refreshed with new medium in a 1:50 ratio and further cultured until reaching an optical density at 600 nm (OD600) of 0.6-0.8.Protein production was induced using 1 mM isopropyl-β-D-1-thiogalactopyranoside (Carl Roth, catalog no.CN08.3).The His6-AP-2β appendage domain, GST-Eps15, and free GST-tag were produced at 37°C for 2 h, and GST-p150 Glued fragments were produced at 21°C overnight.For protein purification, cultures were centrifuged at 4,500  g for 10 min, and the pellet was resuspended in lysis buffer (50 mM Tris [pH, 8.0], 150 mM NaCl, 0.1% Triton X-100, and protease inhibitors).For all subsequent stages, lysates were kept on ice.After resuspension, the cells were lysed by sonication in a Sonics VCX130 PB sonicator (Vibra-Cell) in two 30-s sessions with a 70% amplitude.After sonication, lysates were centrifuged at 13,000  g for 5 min to remove the insoluble fraction.
For the pull-down experiment, the bait protein was then added to Glutathione-Sepharose 4B resin (Merck, catalog no.GE17-0756-01) at a ratio of 30 μl beads for each 10 ml of original bacterial culture and incubated for 1 h with end-to-end rotation.After incubation, the resin was washed three times with lysis buffer, and the prey protein lysate was added.After another 1 h of incubation, the resin was again washed three times with lysis buffer and prepared for Western blot by dissolving in 1 Laemmli buffer and incubation in a heat block at 94°C for 15 min.

Whole-cell lysate preparation
For the Western blot analysis of proteins in whole-cell lysates, Rat-2 cells were lysed in lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 2 mM ethylenediamine tetraacetic acid [EDTA], 0.5% Triton X-100, 0.5% NP-40, 2 mM MgCl2, and 10% glycerol supplemented with protease and phosphatase inhibitors).The total protein concentration in whole-cell lysates was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.Afterward, 4 Laemmli sample buffer was added to the lysates, followed by boiling for 5 min at 95°C.

Immunoprecipitation
For the immunoprecipitation (IP) of endogenous proteins from HEK293T cells, the cells were lysed for 15 min on ice in lysis buffer (20 mM HEPES [pH 7.5], 120 mM KCl, and 0.3%

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS] supplemented with
protease and phosphatase inhibitors, in addition to 100 nM rapamycin for variants in which cells were treated with rapamycin) and spun.Next, 2 µg of anti-AP-2β or anti-p150 Glued primary antibody or IgG (as a negative control) was added to the supernatant and incubated overnight at 4°C while rotating.The next day, a mixture of lysate and antibodies was added to 30 µl of Dynabeads Protein G (Thermo Fisher, catalog no.10004D) and incubated for 4 h at 4°C while rotating.Beads were then washed four times in wash buffer (20 mM HEPES [pH 7.5], 120 mM KCl, and 0.1% CHAPS), eluted in 2 Laemmli sample buffer, incubated for 5 min in 95°C and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot.The IP of AP-2β from rat brain extracts was performed as described previously [2].For the IP of heterologous proteins, HEK293T cells were transfected with pEGFPC1-Ap2b1, GFP-β-actin-p150 Glued , or pEGFPC1 (as a negative control).Forty-eight hours after transfection, the cells were lysed for 15 min on ice in lysis buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, and 0.15 % CHAPS supplemented with protease and phosphatase inhibitors) and spun using a benchtop centrifuge at maximum speed for 15 min at 4°C.The lysates were then incubated with GFP-Trap Agarose (20 µl per variant; Chromotek, catalog no.gta-10) for 2 h at room temperature, followed by four washes with wash buffer (10 mM Tris [pH 7.5], 150 mM NaCl, and 0.5 mM EDTA).The immunoprecipitated proteins were next used for the mTOR kinase assay.

Avi-tag pull down of biotinylated proteins and AP-2β-ear binding
The His6-AP-2β appendage domain was produced as described in the Protein production in bacteria section.For protein purification, cultures were centrifuged at 4,500  g for 10 min, and the pellet was resuspended in lysis buffer (50 mM NaH2PO4/Na2HPO4 [pH 8], 300 mM NaCl, 10 mM imidazole, 10% glycerol, 10 mM β-mercaptoethanol, 0.1% Triton X-100, and protease inhibitors).For all subsequent stages, lysates were kept on ice.After resuspension, the cells were lysed by sonication in a Sonics VCX130 PB sonicator (Vibra-Cell) in two 25-s sessions with a 70% amplitude.After sonication, the lysates were centrifuged at 13,000  g for 5 min to remove the insoluble fraction.The column with 5 ml agarose -Ni -NTA used for affinity chromatography of recombinant protein was equilibrated with 20 ml of lysis buffer and a flow rate of approximately 1 ml/min.The cell lysate that was obtained from 2 L of culture was then added to the column at a flow rate of approximately 0.5 ml/min.After the lysate passed through the column, the agarose resin was washed successively with 50 ml of appropriate buffers: lysis buffer, lysis buffer supplemented with 2 M NaCl, and lysis buffer supplemented with 20 mM imidazole.The protein was then eluted with 25 ml of elution buffer (50 mM NaH2PO4/Na2HPO4 [pH 8], 2 M NaCl, 250 mM imidazole, 10% glycerol, 10 mM βmercaptoethanol 0.1% Triton X-100).One milliliter fractions were collected and verified for the presence of purified protein.Those that contained the highest amount of the His6-AP-2β ear domain were pooled and used for the binding experiments.M-280 streptavidin Dynabeads (Thermo Fisher, catalog no.11205D) were incubated for 1 h with blocking buffer (20 mM HEPES [pH 7.5], 120 mM KCl, 0.5 mg/ml BSA, and 20% glycerol).The beads were washed with lysis buffer (20 mM HEPES [pH 7.5], 120 mM KCl, and 0.3% CHAPS supplemented with protease and phosphatase inhibitors).Forty-eight hours after transfection with BirA and plasmids that encoded Avi-tagged proteins, HEK293T cells were lysed for 15 min on ice in lysis buffer and spun using a benchtop centrifuge at maximum speed (15 min, 4°C).The supernatant was added to the previously prepared M-280 streptavidin Dynabeads and incubated together for 1 h at 4°C while rotating.The beads were then washed on ice four times in wash buffer (20 mM HEPES [pH 7.5], 500 mM KCl, and 0.1% CHAPS) and incubated with 0.2 mg/ml His6-AP-2β ear domain purified from bacteria in binding buffer (20 mM HEPES [pH 7.5], 300 mM KCl, and 0.1% CHAPS) for 2 h at 4°C while rotating.The beads were then washed four times with binding buffer, eluted in 2 Laemmli sample buffer, incubated for 5 min in 95°C and analyzed by SDS-PAGE, followed by Western blot.

Western blot
Protein samples were analyzed by SDS-PAGE and immunoblotting according to standard laboratory protocols.Primary antibodies that were used for Western blot are listed in Table 1.Proteins of interest were detected using HRP-or IRdye-conjugated secondary antibodies in a chemo-luminescence reaction or with the Odyssey LiCor Biosciences system, respectively.The densitometry analysis of the amount of LC3B was performed using Image Studio Lite (LiCor Biosciences) and a previously described method [3].Specifically, the signal intensities for LC3B I and LC3B II were first measured.Based on these values, normalization was performed.First, for all variants in a single replicate, the sum of intensities for a given protein was calculated.Second, individual protein band intensities were divided by this sum.
Such normalization was performed for LC3B I and LC3B II.Finally, after normalizing the values, the ratio between LC3B II and LC3B I was calculated by dividing the normalized values of LC3B II by LC3B I.

Kinase assays
An in vitro phosphorylation assay was performed for 20 min at 30°C in kinase assay reaction buffer (25 mM HEPES [pH 7.5], 50 mM KCl, and 10 mM MgCl2) in the presence of 2.5 μCi [γ-33 P]ATP (Hartmann Analytic) and 2.5 mM "cold" ATP using a recombinant, active mTOR kinase fragment (1362-2549 aa; Millipore, catalog no.14-770) and proteins that were immunoprecipitated from HEK293T cells or commercial mTOR substrate (Merck Millipore, catalog no.12-645).The reaction was stopped by adding 4 Laemmli sample buffer and boiling for 5 min at 95°C.Next, the samples were separated by SDS-PAGE.The gel was stained with Coomassie Brilliant Blue and dried.The radioactive signal was detected and analyzed using a Typhoon Trio+ Phosphorimager (GE Healthcare).Western blot analysis of endogenous p150 Glued , Arp1, p62, S6, and P-S6 (Ser235/236) levels in HEK293T cells and co-immunoprecipitation of p150 Glued , Arp1, and p62 from HEK293T cells that were treated for 2 h with 0.1% DMSO, 100 nM rapamycin (RAPA), or 100 nM AZD80550.Shown is a representative example from N = 2 independent experiments.

Fig. S11 .
Fig. S11.(A) Representative images of Rat2 cells that were treated for 2 h as indicated, with

Fig. S14 .
Fig. S14.Results of analysis of lysotracker staining distribution and intensity in cells

Fig. S15 .
Fig. S15.Blot transparency.Full-size blots that correspond to cropped images that are